KR100218855B1 - Chimeric gene for herbicidal resistance - Google Patents

Abstract

New chimeric gene (I) confers resistance to herbicides (II) of the 3,5-dihalo-4-hydroxybenzonitrile type comprises: (i) at least one gene (Ia) encoding resistance to (II); (ii) a heterologous promoter; and optionally(iii) a polyadenylation signal. The promoter is from a gene naturally expressed in plant cells: (a) the 35S RNA promoter of cauliflower mosaic virus (P1); or (b) the promoter from the small subunit of the ribulose-1,5-bis-phosphatocarboxylase oxidase (Rubisco) gene of Helianthus annuus (P2). Also claimed are: (1) vectors used to transform plants to make them resistant to (II); (2) microorganisms comprising (I); (3) plant cells comprising (Ia); (4) transformed plants (or plant parts) obtained by regenerating (3); (5) seeds of (4); and (6) a process of selectively weeding using (II) applied to (4).

Description

Chimeric Genes for Herbicide Tolerance

The present invention provides a chimeric gene that can be used to confer resistance to a herbicide based on 3,5-dihalo-4-hydroxybenzonitrile, a method of transforming plant cells using the gene, and regeneration in these cells. To transgenic plants.

Incorporation of genes encoding nitrilas specific for the degradation of these types of herbicides into the genome of plants results in the plants being able to produce these herbicides, in particular 3,5-dibromo-4-hydroxybenzonitrile or bromine. It is well known from European Application No. 229,042 to have resistance to moxinil. This technique has useful results, but in order to increase its chances of success and increase its economic potential, especially with respect to the level of expression in plants and, therefore, the quality of the plant's resistance to the herbicides. This is necessary.

In the present technology, "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, and "plant cells" are derived from plants and not differentiated, such as callus or embryo. It is understood to mean any cell that is capable of forming a tissue or part of a plant or differentiated tissue or seed such as a plant.

The object of the present invention is to meet this need.

It is at least one gene encoding a nitriase resistant to the herbicide, the foreign promoter, which can be used to confer plants to herbicides based on 3, 5-dihalo-4-hydroxybenzonitrile. And, in some cases, to a chimeric gene containing a polyadenylation signal site (wherein the promoter is derived from a gene that is naturally expressed in plant cells and is a promoter of 35 S RNA (CaMV 35S) of cauliflower mosaic virus). , Sunflower (Helianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) small subunit (SSU) promoter.

1 shows the remodeling of T DNA that possesses the right and left borders of pTiA6 in their original directions.

Figure 2 shows the introduction of T DNA into pBR 322.

Figure 3 shows the cloning of genes for resistance to gentamicin.

4 shows the cloning of genes for resistance to kanamycin.

The promoter of the chimeric gene according to the present invention is naturally expressed in a plant, that is, a viral type such as 35 S RNA (CaMV 35S) of a non-plant type, for example, cauliflower mosaic virus, or preferably monocot or dicotyledonous plant. Plant type, particularly from the gene of the small subunit of sunflower (Hlianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO). It is possible to use these promoters alone or in combination. The type of selection depends on the nature of the plant to be transformed (monolithic or dicotyledonous). Therefore, it is better to use the small subunit RubisCO of sunflower for transformation of dicotyledonous plants.

Each of these promoters can be obtained as follows: 1) Promoter of Cauliflower Mosaic Virus 35 S RNA (CaMV 35S): Isolation of this promoter has been described by Odell et al. (1985). A clone (pJO5-2) comprising about 850 bp upwards at the transcription initiation site was described as selected for construction. The EcoRI-HindIII fragment was isolated and its ends blunted with Klenow polymerase and cloned into the HincII site of vector pUC19 (Yannish-Perron et al., 1985). It was. This clone was digested by the action of Xbal and PstI, and the resulting fragments were treated with phage T4 polymerase to make their ends blunt. This fragment was cloned into pUC19 Cm (Buckley, 1985) digested with SmaI and XbaI and treated with Klenow polymerase. The clone thus obtained was represented by pRPA-BL I45. By treating the 3 ′ terminal AccI site with Klenow polymerase and linking it with an EcoRI site treated with Klenow polymerase of fragments located downstream from the promoter, the EcoRI site is reformed and the resulting sequence is transcribed. Starting at the initiation site,

2) Promoter of small subunit of Sunflower (Helianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO).

The gene from which this promoter is derived was isolated by Waxman et al. (1987). The EcoRI fragment containing the promoter of this gene was cloned into the 3 'portion of the promoter just above the mp 18, polylinker of this vector. This clone was then linearized with BstXI and treated with Ba131 exonuclease. The mixture of fragments thus obtained was treated with SalI followed by Klenow polymerase and finally linked at low DNA concentrations. The clones obtained after this manipulation were sequenced and one of them with the subsequent sequence below the putative transcription initiation site was selected:

The C1aI linker (A T C G A T) was introduced at the PstI site of this clone. Thus, by treating this C1aI site with Klenow polymerase and linking it with an EcoRI site treated with Klenow polymerase of fragments located underneath this promoter, the EcoRI site is reformed and the resulting sequence is estimated Starting at the site of transcription initiation,

According to another aspect of the invention, the chimeric gene contains a non-translating intermediate site (linker) between the coding gene and the promoter, which may be selected from the group comprising: , A linker of pUC 19 partially modified by cloning and having the following sequence:

On the other hand, the untranslated site of a small subunit of maize RubisCO: it comes from the cDNA corresponding to the gene described by Lebrun et al. (1987). It is an EcoRI-NcoI fragment having the following sequence:

On the other hand, the untranslated region of the small subunit of sunflower RubisCO: this region comes from cDNA separated by Waxman and Freysinet (1987). It has never been isolated as such and is always found preceding the transport peptide of the sunflower RubisCO. The sequence is as follows:

The chimeric gene according to the present invention optionally contains a polyadenylation region or site, which may be, for example:

1) The polyadenylation site of the nopaline synthase gene (Bevan et al., 1983) of pTi 37. This site is a 260-bp MboI fragment cloned into the SmaI site of M13mp18 and treated with Klenow polymerase to introduce BamHI and EcoRI sites at the 5 'and 3' ends, respectively (Fraley et al., 1983). And patent application PCT No. 84/02913. The BamHI site was cloned at the Sall site, treated with Klenow polymerase of pUC 19 by treatment with Vigna radiata nuclease. This fragment now contains a HindIII site at the 5 'end, which can be linked at the 3' end of the nitrile gene.

2) Polyadenylation site of a small subunit of maize RubisCO: This site was isolated in the form of a 540-bp Smal-Bg1II fragment of the gene described by Revrun et al. (1987). C1I linker (ATCGAT) was introduced at the Smal site. After cleavage with ClaI and filling with Klenow polymerase, the fragment was cloned into pUC19 digested with PstI, treated with phage T4 polymerase and cut back with BamHI. This operation allowed the HindIII site to be introduced at the 5 'end of the polyadenylation site. The sequence obtained is as follows:

According to another aspect of the invention, the chimeric gene is optionally with a site encoding the small subunit of maize RubisCO transport peptide and the small subunit of sunflower between the intermediate region and the nitrile gene. It may be good to include it, which is chosen from the group that was made up. The function of the transport peptide in natural genes is to allow small subunits of RubisCO to enter the chloroplast stroma.

They should likewise enter nitrilease into this compartment when introduced between the intermediate region described above and the structural gene of the nitrilease:

Transport peptides for small subunits of maize RubisCO: This fragment comes from the cDNA corresponding to the gene described by Revrun et al. (1987). It is a 141-bp NcoI-SphI fragment, which spans the SphI site, which is the cleavage site of the translation initiation codon and the transport peptide. By treating the SphI terminus of this fragment with phage T4 polymerase and linking it with the NcoI terminus treated with the Klenau polymerase of the nitrilase gene, it allows for the production in the stromal of the chloroplasts of the unmodified nitrilase. The sequence is reshaped.

2) Transport peptide of small subunit of sunflower RubisCO: This fragment comes from cDNA isolated from Waxman and Freycinet (1987). This sequence does not originally have a SphI site at the cleavage site of the transport peptide. The sequence of this region is as follows:

One C was replaced with an A starred by a control anthropogenic mutation, thereby creating a SphI site. To carry out this manipulation, the method of Zoler and Smith (1984) was used. 270-bp EcoRI-SalI fragment was cloned into M13mp19am4. Derived from M13mp19, the vector has an amber mutation of gene 4 at base 5327 and cannot propagate in a strain that does not have an inhibitor against this type of covariation. After single-stranded purification of the recombinant phage, three oligonucleotides were hybridized in one step. The sequence of these phosphorylated oligonucleotides is as follows:

They each enable the following:

1: mutation of the fragment at the cleavage site of the transport peptide,

2: modification of amber mutation,

3: priming of the top sequence of an anthropogenic fragment.

After simultaneous action of Klenau polymerase and phage T4 ligase in the presence of four kinds of nucleotides, the resulting mixture was transformed into HB2154 strains and then plated on the lawn of HB2151 [Carter ), 1985]. Of the clones obtained, those with additional SphI sites were sequenced to confirm this structure and one of them was used for the preparation of the chimeric gene. In the region of the cleavage site of the transport peptide, this sequence is now as follows:

This fragment is used in the same manner as was used for the fragment encoding the small peptide transport peptide of maize RubisCO.

The assembling of the chimeric genes is done according to the schemes of Figs. The resulting genes were placed in one or two types of vectors, each of which was assigned a reference number. The various vectors thus prepared are described in Table 1 below:

Vectors used: Various camera configurations, resulting in the vectors pRPA-BL-142 and pRPA-BL-150Aalphal shown in FIGS. 1-4, were introduced into the plant by the Agrobacterium tumefaciens transport system. It became. Carry vectors created for this purpose have the following characteristics:

-replication and transport origin from pBR 322,-genes for resistance to bacteria, eg, gene resistance to gentamicin,-COS sites from phage lambda,-both right and left borders of pTiA6 T DNA, -Optionally, resistance to a selection gene of nucleated cells, such as kanamycin, and the like-optionally, a fragment containing the 1ac alpha compensation gene of pUC18.

Configuration of pRPA-BL-142 (FIGS. 1 to 4): The right and left borders (FIG. 1) of the left T DNA of pTiA6 were first subcloned:-Right border: Barfker et al. (1983) The BaMHI-EcoRI fragment extending from 13774 to 16202 in the numbering system of) was cloned into the pGEM 1 (Promega Biotech Co., Ltd.) at the sites, to obtain pBL-17. This plasmid was digested with NruI (No. 14276 and No. 14475) and EcoRI (No. 16202) and treated with Klenow polymerase. By linking this NruI site with the filled EcoRI site, the EcoRI site was regenerated at 14276 to obtain plasmid pBL-19.

Left border: Cloning the stretched HindIII fragments from 602 to 3390 in a Barker et al. (1983) system into the corresponding site of pGEM 1 to obtain pBL-21, where the left border is on the opposite side of the polylinker. have. This plasmid was digested with Acc 1 (1161 and 2687), treated with Klenow polymerase and linked. The resulting plasmid pBL-26 contains fragments extending from No. 602 to No. 1161 inserted between the HindIII and Xbal sites.

Preparation of T DNA: By introducing EcoRI-BamHI fragment of pBL-19 into the corresponding site of pBL-26, T DNA having right and left borders in the original direction of pTiA6 was reshaped. The resulting plasmid is represented by pBL-70.

Introduction of T DNA into pBR 322 (Figure 2): After pBL-70 was cut with HindIII, this site was treated with Klenow polymerase and the plasmid was digested again with EcoRI. The resulting fragment was cloned into pBR 322 digested with PruII-EcoRI. The resulting clone is represented by pRPA-BL-112.

Cloning of genes resistant to gentamicin (Figure 3): Genes resistant to gentamicin were obtained from pPH1 J1I (Hirsh and Bringer, 1984). This plasmid was digested with BamHI and HindIII and all of the fragments were cloned into pUC 19 digested with the same enzymes. After screening for ampicillin + gentamycin, clones containing 2.45-kbp fragments were isolated. The clone selected for subsequent manipulation was named pRPA-BL-133. At the BamHI site of this clone, a 1.6-kb BalII fragment isolated from pHC 79 (Hohn and Collins, 1980) and containing the COS site of phage lambda was introduced. Two fragments, pRPA-BL-134 and pRPA-BL-135, could be obtained by this fragment inserted in both directions.

Preparation of Integral Vectors (FIG. 3): To combine the various other parts described above in one and the same vector, plusmid pRPA-BL-134 and pRPA-BL-135 were digested with SmaI and HindIII, and were treated for gentamicin. Inserts containing the gene of resistance and COS sites of phage lambda were treated with Klenow polymerase. Plasmid pRPA-BL-112 was digested with PstI and EcoRI and treated with phage T3 polymerase. Both fragments were linked and clones containing both resistance to gentamicin, COS site, T DNA and origin of replication of pBR 322 were selected. PRPA-BL-141 and pRPA-BL-142 were obtained from plasmid pRPA-BL-134, and pRPA-BL-143 and pRPA-BL-144 were obtained from pRPA-BL-135. PRPB-BL-142 was chosen for the introduction of chimeric genes to be transported into plants. From this vector, the constitution (FIG. 4) containing the marker gene NOS-NPTII-NOS was obtained. Plasmid pRPA-BL-142 was digested with XbaI and its ends were reduced by the action of Vigna radiata nuclease. Separately, pEND4 K (Klee et al., 1985) was digested with EcoRI and treated with Klenow plymerase. A fragment of 1.6-kbp containing the chimeric gene of resistance to kanamycin was isolated and introduced into pRPB-BL-142. One product of these fusions was named pRPB-BL-150A and was selected for later manipulation. In order to facilitate cloning into this vector, a HagII fragment treated with phage T4 polymerase and containing a lac alpha compensation gene isolated from pUC18 (Jannish-Peron et al., 1985) was obtained by vigna radiata nu. It was introduced at the BamHI site treated with clease. The obtained vectors were named pRPA-BL-150Aalphal and pRPA-BL-150Aalpha2. pRPA-BL-150Aalphal was a vector used as a mood for the introduction of genes into plants.

Use of pRPA-BL-142 and pRPA-VL-150Aalphal

These vectors are not maintained in Agrobacterium by themselves. In order for them to be retained, they must be integrated by simple recombination in the plasmid residing in this bacterium. This may occur via one of the fragments, such as a site present in cosmids such as pVK 102, or a fragment of pBR322 for plasmids having the sequences. This is true for the Ti plasmid (Zambryski et al., 1983) of the GV3850 strain, which is also a host of pRPA-BL-142 and pRPA-BL-150Aalphal. Using the origin of replication of pBR322, these plasmids are transferred into Agrobacterium through a three-part system described by Ditte et al. (1980).

Transformation of plant components

To test the efficacy of these chimeric genes, the latter were transferred into components according to the method described below:

A-Transformation Method

1. Tobacco

The vector is introduced into non-carcinogenic strain Agrobacterium EHA 101 (Hood et al., 1987) with cosmid pTVK 291 (Komari et al., 1986). The transformation technique is based on the method of Hoch et al. (1985). The regeneration method of commercial tobacco PBD6 (Source: SEITA, France) is described below.

Regeneration of tobacco PBD6 in the in vitro culture of the leaves is carried out on Murashige and Squag (MS) basal medium containing 30 g / 1 sucrose. In vitro cultures of leaves are taken from plants grown in greenhouses or in vitro and transformed according to the three consecutive stages of the leaf disc technique (Science 1985, Vol. 227, pp. 1229-1231).

의 NAA 및 2

/1의 BAP를 함유하는 MS+30g 수크로오스상에서 15일동안 새싹을 유도하는 것으로 이루어져 있다.The first step is 0.05

NAA and 2

Induce buds for 15 days on MS + 30 g sucrose containing BAP of / 1.

The second step is to develop the shoots formed during the first step: it is carried out for 10 days on MS + 30 g / 1 sucrose containing no hormones.

The third step is to root each shoot. The rooting medium contains salts, vitamins and sugars and is diluted twice-fold. It contains no hormones at all. After 10-15 days, the transplanted shoots are transferred to the ground.

Determination of Hormone Balance

/1와 NAA 0.05 및 0.1

/1 에 대하여 관찰한 25개의 호르몬 밸런스 BAP 0.2-0.5-1-1.5.2 및 NAA 0-0.5-0.05를 시험함으로써 얻어졌다. 0.05 NAA와 2

/1 BAP의 혼합물이 채택되다.The optimal refresh rate is BAP 1.5 and 2

/ 1 and NAA 0.05 and 0.1

It was obtained by testing the 25 hormone balances BAP 0.2-0.5-1-1.5.2 and NAA 0-0.5-0.05 observed for / 1. 0.05 NAA and 2

A mixture of 1 BAP is adopted.

2. Other Dicotyledonous Plants

The plant components as indicated in Table 2 were used to transform the dicotyledonous plants shown in Table 2 using Agrobacterium A281 of a carcinogenic strain carrying cosmid pTVK291 linked to the vector.

Determination of resistance to B-bromoxynil

1. Tobacco

/1)의 존재하의 칼루스들의 성장에 의해서, 그리고 생체 내의 경우 오픈-필드 처리에 추천되는 용량의 10 배를 나타내는 용량으로 잎에 브로목시닐 또는 이옥시닐을 살포하는 방법으로 측정하였다. 얻어진 결과는 표 1a에 요약되어 있다.Resistance in vitro is octanoate bromoxynil (20)

The growth of callus in the presence of / 1) and, in vivo, was measured by spraying bromoxynil or oxynyl on the leaves at a dose representing 10 times the dose recommended for open-field treatment. The results obtained are summarized in Table 1a.

2. Other Dicotyledonous Plants

의(옥타노에이트형의) 브로목시닐의 존재하에 칼루스들의 성장으로 측정하였다. 모든 경우들에 있어, 결과는 양성이다(표 2).Resistance is 10 per liter of medium in vitro

It was measured by the growth of callus in the presence of (octanoate type) bromoxynil. In all cases, the result is positive (Table 2).

The chimeric gene of the present invention enhances the level of expression in plants and the quality of the plant's resistance to the herbicides, and is therefore economically advantageous.

Claims (4)

Genes encoding nitriases specific for degradation of the herbicide, linked to functional foreign promoters and polyadenylation signal sites, which confer resistance to herbicides based on 3,5-dihalo-4-hydroxybenzonitrile A chimeric gene encoding a nitrile derived from bacteria, wherein the promoter is derived from a gene that is naturally expressed in plant cells and functionally with the gene of 35 S RNA (CaMV 35S) of cauliflower mosaic virus. Linked promoter, promoter of small subunit (SSU) of sunflower (Hlianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) and 35 S RNA of cauliflower mosaic virus (CaMV 35S) Promoter of sunflower (Helianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) small subunit (SSU) Useful for transforming a plant into a plant that is resistant to the herbicide. The vector of claim 1 consisting of plasmids. Genes encoding nitriases specific for degradation of the herbicide, linked to functional foreign promoters and polyadenylation signal sites, which confer resistance to herbicides based on 3,5-dihalo-4-hydroxybenzonitrile Chimeric gene encoding a nitrile derived from bacteria, wherein the promoter is derived from a gene that is naturally expressed in plant cells and functionally with the gene of 35 S RNA (CaMV 35S) of cauliflower mosaic virus. Linked promoter, promoter of small subunit (SSU) of sunflower (Hlianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) and 35 S RNA of cauliflower mosaic virus (CaMV 35S) Promoter of Small Subunit of Sunflower (Helianthus annuus) Ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) Microorganisms). Genes encoding nitriases specific for degradation of the herbicide, linked to functional foreign promoters and polyadenylation signal sites, which confer resistance to herbicides based on 3,5-dihalo-4-hydroxybenzonitrile Chimeric gene encoding a nitrile derived from bacteria, wherein the promoter is derived from a gene that is naturally expressed in plant cells and functionally with the gene of 35 S RNA (CaMV 35S) of cauliflower mosaic virus. Linked promoter, promoter of small subunit (SSU) of sunflower (Hlianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) and 35 S RNA of cauliflower mosaic virus (CaMV 35S) Promoter of sunflower (Helianthus annuus) ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) small subunit (SSU) Plant cells containing genes resistant to the herbicides, obtained by integration of the herbicide.

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